In vivo inhibition of keratinocyte growth factor receptor expression by estrogen and antagonism by progesterone in the mouse mammary gland.

Mammary gland development is regulated by complex interactions among mammogenic hormones and locally derived paracrine growth factors. In epithelial tissues, keratinocyte growth factor (KGF or FGF-7) originates in the stroma while its receptor (KGFR or FGFR2-IIIb) is present only in the epithelium. Previous work showed that estrogen but not progesterone could stimulate the synthesis of KGF in mammary stroma in vivo. The effects of 17 beta-estradiol and progesterone on KGFR expression in vivo were examined in these studies. Peripubertal and mature virgin mice received subcutaneous injections of hormone in sesame oil after which KGFR mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. Estradiol treatment caused a dose- and time-dependent decrease in KGFR mRNA level in mice from both age groups while stimulating ductal growth after 7 days of treatment. Inhibition of KGFR expression was near maximal at an estradiol dose of 2 microg after 1 day of treatment. Progesterone injection increased KGFR mRNA levels but this effect correlated with the stimulation of ductal growth. However, when progesterone was co-administered with estradiol, KGFR mRNA levels were maintained in the absence of any effect on ductal growth. Thus, estradiol inhibited KGFR mRNA only when elevated unopposed by progesterone. These data show that KGFR expression is determined by the ratio of estradiol and progesterone and suggests a mechanism through which these hormones can co-operate to optimize their growth-promoting effects. Consequences of hormone imbalance are also implicated.

[Ramatroban (Baynas): a review of its pharmacological and clinical profile].

Bayer has been interested in the observations that metabolites of arachidonic acid are involved in allergy and inflammation. Ramatroban was thus developed as a therapeutic agent for allergic and inflammatory diseases. Ramatroban showed an antagonistic action on the thromboxane A2 (TXA2) receptor in in vitro experiments using platelets or arteries. It inhibited the permeability of capillary and also the infiltration of eosinophils in nasal mucosa. Ramatroban had an inhibitory effect on the nasal resistance stimulated by either U-46619 or antigen challenge in in vivo experiments. The concentration of nasal TXA2 was increased when the antigen was challenged to allergic patients. Clinical trials demonstrated that ramatroban decreased sneezing, rhinorrhea, and rhinostenosis in patients enrolled in the study. No serious adverse reaction of ramatroban was observed in patients throughout the trials. The treatment with ramatroban is safe and improves nasal symptoms.

Pattern of expression of the KGF receptor and its ligands KGF and FGF-10 during postnatal mouse mammary gland development.

The expression of the KGF receptor (KGFR) and its stromal ligands, KGF and FGF-10, was compared during mouse mammary gland development. KGFR expression in mammary parenchyma is maximal in mature virgin mice, declines during pregnancy and lactation, but rises after weaning. The rise in KGFR mRNA in the virgin animal corresponds to parenchymal growth. The fall in KGFR expression in pregnancy is driven by hormone-induced alveolar differentiation since the level of KGFR mRNA is 5-fold higher in isolated ductal cells compared to alveolar cells. KGF and FGF-10 expression patterns differ during ductal development. FGF-10 is also expressed at about a 15-fold higher molar level than KGF. During pregnancy and lactation, expression of KGF and FGF-10 decreases in intact fat pads but is unchanged in parenchyma-free fat pads. Thus, the decrease in KGF and FGF-10 expression observed in intact glands during pregnancy and lactation is not a direct consequence of the changing hormonal milieu but more likely reflects an increase in the ratio of epithelium to stroma. Differences in the level and pattern of expression of mRNA for KGF, FGF-10, and the KGFR during postnatal development of the mouse mammary gland are a result of morphological development, changes in the ratio of stroma to epithelium, and hormonal regulation of cell differentiation. These changes suggest that the biological roles that these growth factors play are regulated by fluctuations in both growth factor and growth factor receptor expression and that KGF and FGF-10 may have different regulatory functions.

Attenuation and loss of hormonal modulation of KGF (FGF-7)/KGF receptor expression and mitogenesis during mammary tumor progression.

Keratinocyte growth factor (KGF), alone and in synergism with progesterone (P) and prolactin (PRL), is mitogenic for normal mammary epithelium (ME) in vitro. In addition, P can upregulate ME sensitivity to KGF by slowing KGF receptor (KGFR) mRNA turnover in vitro. These hormonal interactions with KGF in vitro raise the possibility that alterations in these interactions can play a role in hormone-dependent mammary tumor growth and progression. The effect of hormones on KGF mitogenesis and the regulation of KGFR expression was examined in pregnancy-dependent (PDT) and ovarian-independent (OIT) mouse mammary tumors. In serum-free, collagen gel cell culture, dose/response (2-20 ng/ml) and time course studies showed that KGF stimulated the proliferation of PDT (not OIT) cells but synergism with P or PRL was not observed. The level of KGFR mRNA in PDT cells was not significantly different from normal ME but in OIT it was reduced more than 90%. P did not affect KGFR mRNA turnover in cultured PDT cells. However, KGFR mRNA was more stable in PDT cells compared to normal ME; after 6 days culture in basal medium, KGFR mRNA levels declined 40% vs. 85% previously shown for normal ME. Determination of KGF mRNA levels in tissues showed that it was lower in PDT compared to normal mammary gland and not detectable in OIT. These data show that in PDT both KGF-stimulated mitogenesis and the regulation of KGFR expression are independent of hormones. OIT has progressed to independence from any KGF influence. Thus, a subset of hormonally regulated pathways related to epithelial/stromal cell interactions can be lost in hormone-dependent mammary tumors during tumor progression.

Estrogen treatment in vivo increases keratinocyte growth factor expression in the mammary gland.

Proliferation and differentiation of mammary epithelia are regulated by the combined action of systemic hormones and locally derived paracrine growth factors. Keratinocyte growth factor (KGF) is a potential candidate stromal factor that may participate in the hormonal control of stromal/epithelial interactions. In this study, we have examined the in vivo effect of 17beta-estradiol (E) treatment on KGF expression in mammary glands of peripubertal (5-week-old) and mature (11-week-old) mice. Mice received subcutaneous injections of hormone after which KGF mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. E treatment caused a dose- and time-dependent increase in KGF mRNA levels in intact mice from both age groups. Neither 17alpha-estradiol nor progesterone injection affected KGF mRNA levels. Comparison of the relative expression of KGF in parenchyma-free fat pads and in intact glands demonstrated that the basal and E-dependent KGF mRNA levels did not require the presence of mammary epithelium. ELISA assay of KGF tissue content demonstrated that concomitantly with an up-regulation of mRNA, E treatment also increased KGF protein in mammary glands from peripubertal and mature mice. These data show that E treatment stimulates both KGF mRNA and protein expression in mammary stroma in vivo and raises the possibility that KGF has a role in E-regulated mammary gland development.

Altered MAP kinase (ERK1,2) regulation in primary cultures of mammary tumor cells: elevated basal activity and sustained response to EGF.

An elevation in total MAP kinase activity and expression has been observed in breast cancer tissue. However, the mechanisms underlying these changes in kinase activity and regulation by growth factors are not well characterized. In these studies, the effect of the potent mammary mitogen, epidermal growth factor (EGF), on the activation of the mitogen-activated protein kinases, ERK1 and ERK2 (extracellular regulated protein kinases 1 and 2, respectively), was compared in primary cultures of normal mouse mammary epithelial cells and in a hormone-responsive mouse mammary tumor. In normal epithelium, EGF stimulated an early rise in ERK activity at 4 min followed by a rapid decline, whereas a sustained (1 h) elevation of ERK activity was observed in the tumor cells. The time course of ERK activity in both cell types coincided with the phosphorylation state of the EGF receptor, suggesting that altered regulation of EGF receptor phosphorylation or EGF receptor turnover produces an enhanced ERK response to EGF in tumor cells. The MEK inhibitor, PD 098059 inhibited EGF-stimulated proliferation and ERK activity in a parallel, dose-dependent manner showing that ERK activation is at least permissive for the proliferative response to EGF. In addition, tumor cells showed a 4-fold elevation in basal (or ligand-independent) activity over normal cells without an increase in total enzyme level, and a preferential activation of ERK1 by EGF. These EGF-dependent and -independent changes in ERK regulation in the hormone-responsive mammary tumor underscore how multiple alterations in the regulation of this pathway may play a role in mammary tumorigenesis.

Mammogenic hormones differentially modulate keratinocyte growth factor (KGF)-induced proliferation and KGF receptor expression in cultured mouse mammary gland epithelium.

Stromally derived keratinocyte growth factor (KGF) can play an important role in mammary gland development as a mesenchymal/stromal mediator of epithelial growth and morphogenesis. However, the possible coordinate regulation of mammary gland development by mammogenic hormones and KGF is unexplored. In these studies, the direct effect of mammogenic hormones on KGF-mediated mammary epithelial mitogenesis and expression of the KGF receptor was examined using primary mouse mammary epithelium growing in serum-free, collagen gel cell culture. Addition of KGF produced an average 7-fold increase in cell number after 10 days of culture. This effect of KGF was further increased in the presence of PRL (9-fold) or progesterone (P; 15-fold), with the combination of P and PRL (22-fold) producing the strongest synergistic stimulation. Estrogen did not show any additional stimulation of growth either alone or in combination with PRL and/or P. Ribonuclease protection analysis showed that epithelial cells grown in medium supplemented with P, but not PRL or estrogen, exhibited a 10-fold higher steady state level of KGF receptor (KGFR) messenger RNA (mRNA). KGFR expression was not induced by short term P exposure, suggesting an effect on mRNA stability rather than transcriptional activation. Time-course studies showed that an early decrease in the level of KGFR mRNA in basal cultures was significantly reduced by P addition. Measurement of RNA turnover after actinomycin D treatment showed that P increased the t(1/2) of KGFR mRNA compared with basal medium. Thus, P and PRL may differentially potentiate the direct mitogenic effect of KGF: P partly by elevation of the level of KGFR mRNA, and PRL principally by intracellular pathways not affecting KGFR expression.

Rcho-1 trophoblast cell placental lactogens: complementary deoxyribonucleic acids, heterologous expression, and biological activities.

In this report, we have investigated placental lactogens (placental lactogen-I, PL-I; PL-I variant, PL-Iv; PL-II) expressed by differentiated Rcho-1 trophoblast cells. A complementary DNA (cDNA) library to differentiated Rcho-1 trophoblast cells was constructed and screened with probes to detect PL-I and PL-II. Sequence analysis of three independent Rcho-1 PL-I cDNAs indicated that they significantly differed from the previously reported PL-I sequence but more closely resembled a related cDNA referred to as PL-I mosaic (PL-Im). Upon further analysis, Rcho-1 PL-I/PL-Im transcripts could be detected in Rcho-1 trophoblast cells and normal developing placental tissue; however, the previously reported PL-I transcript could not be identified from the same sources. Given these results, we examined the original PL-I cDNA by PCR and nucleotide sequence analyses. The sequence differed from the original report and was found to be identical to the Rcho-1 PL-I and PL-Im cDNA clones. Thus, PL-I, Rcho-1 PL-I, and PL-Im are equivalent and should be referred to as PL-I. The PL-I gene was localized to chromosome 17 of the rat genome, similar to other PRL family members. Rcho-1 PL-II cDNAs were identical to the published PL-II sequence. PL-Iv cDNAs were isolated from differentiated Rcho-1 cells via an RT-PCR strategy and found to be identical to previously isolated PL-Iv cDNAs. Rcho-1 PL-I and PL-II cDNAs were subcloned into the pcDNA3 expression vector and recombinant protein produced in HRP-1 cells. Both recombinant Rcho-1 PL-I and PL-II proteins significantly stimulated the proliferation of lactogen-dependent rat Nb2 lymphoma cells and mouse mammary epithelial cells. In summary, we show that the Rcho-1 PL-I corresponds to PL-Im and Rcho-1 PL-Iv and PL-II are identical to their previously described placental counterparts. Additionally, both recombinant Rcho-1 PL-I and PL-II proteins are biologically active.

Multifunctional phosphatidic acid signaling in mammary epithelial cells: stimulation of phosphoinositide hydrolysis and conversion to diglyceride.

We have shown previously that phosphatidic acid esterified to polyunsaturated fatty acids is mitogenic for primary cultures of mouse mammary epithelial cells embedded within collagen gels. We hypothesized that this mitogenic competence resulted from the ability of this phospholipid to activate multiple signal transduction pathways in mammary epithelium. A closer examination of this hypothesis was undertaken by examining the effect of exogenous phosphatidic acid on phosphoinositide (PI) hydrolysis and its intracellular metabolism to diglyceride, an activator of protein kinase C. For assays of phosphoinositide-specific phospholipase C activation, mammary epithelial cells from virgin Balb/c mice were isolated by collagenase dissociation of mammary glands and cultured on the surface of Type I collagen-coated culture dishes. Phosphatidic acid (PA) stimulated a sustained increase in inositol phosphates and caused inositol phospholipid depletion when added to cells in which inositol phospholipids were prelabeled with 3H-myoinositol. This effect was specific for PA among phospholipids tested. Neither lineoleic acid, that can be released from PA, nor prostaglandin E2 affected PI hydrolysis. When mammary epithelial cells were cultured inside collagen gels in the presence of exogenous PA or phosphatidylcholine (PC) radiolabeled with 3H-glycerol, PA was found to persist intracellularly and be dephosphorylated to diglyceride (an activator of protein kinase C) to a greater extent than PC, a nonmitogenic phospholipid. In contrast to PA, epidermal growth factor (EGF) only slightly stimulated PI hydrolysis, showing that these two different growth-promoting factors do not actively couple to the same signal transduction pathways in mammary epithelial cells. These results show that PA may activate multiple pathways in mammary epithelial cells either directly or via its metabolism to diglyceride.

Role of GTP-binding proteins in the polyunsaturated fatty acid stimulated proliferation of mouse mammary epithelial cells.

Polyunsaturated fatty acids enhance the proliferation of mouse mammary epithelial cells stimulated by epidermal growth factor (EGF) by modulating the post-receptor signaling pathways. The growth stimulatory effect of these fatty acids is completely inhibited by pertussis toxin, whereas the inhibition of EGF and insulin stimulated growth is only partial. The treatment of cell cultures with 12-O-tetradecanoyl-phorbol-13 acetate (TPA) reverses the growth inhibitory effect of pertussis toxin and fully restores the growth as was in the control cultures untreated with the toxin suggesting a role for PKC in this reversal. It appears that the functions of Gi-proteins are required in the mediation of fatty acid effect on growth. The predominant types of Gi alpha in mammary epithelial cells are Gi alpha 1, Gi alpha 2, and Gi alpha 3. Among these, the levels of Gi alpha 1 and 2 appears to be regulated by steroid hormones. Linoleic acid raises the level of GTP-bound Ras in the cells above the levels induced by EGF. Pertussis toxin reduces the level of Ras-GTP and inhibits phosphorylation of MAP kinase by EGF. It has been speculated that Gi-proteins interact with the receptor bound nucleotide exchange factor and the membrane anchored Raf kinase and constitute two sites for pertussis toxin action. The phosphorylation by PKC may uncouple Gi-protein interaction with these effectors and enable the agonist-induced signals to bypass the inhibitory action of PT on growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the proliferative response to lysophosphatidic acid in primary cultures of mammary epithelium: differences between normal and tumor cells.

The effect of lysophosphatidic acid (LPA) on the proliferation of normal and tumor mouse mammary epithelial cells in primary, serum-free, collagen gel cell culture was evaluated. LPA stimulated the growth of normal mammary epithelial cells from mature virgin mice. The growth of pregnancy-dependent tumors (PDT) was generally stimulated, although the response was attenuated in some of these tumors compared to normal cells. In contrast, the growth of 70% of ovarian-independent tumors (OIT) was inhibited by LPA; the remainder were unaffected. LPA stimulated cAMP accumulation and phosphoinositide (PI) hydrolysis in normal, PDT, and OIT. Thus, the regulation of adenylyl cyclase and PI-specific phospholipase C by LPA is similar in normal and tumor cells. Pertussis toxin (PT) partially inhibited LPA-stimulated growth in normal cells but did not affect LPA-stimulated PI hydrolysis or cAMP accumulation. Thus, PT-sensitive and -insensitive proliferative pathways are activated. PT also inhibited LPA-stimulated growth of PDT but generally had no effect on the growth of OIT. These results show that the mitogenic response to LPA is attenuated in the hormone-dependent phenotype and switches to growth inhibition in hormone-independent tumors. Furthermore, LPA stimulates multiple signal transduction pathways mediated by PT-sensitive and -insensitive G proteins. The PT-sensitive pathways are not tightly coupled to the proliferative response to LPA in tumor cells. These data suggest that alterations in G protein function may occur during tumor progression.

Omega-3 and omega-6 fatty acids and PGE2 stimulate the growth of normal but not tumor mouse mammary epithelial cells: evidence for alterations in the signaling pathways in tumor cells.

The direct effect of omega-3 and omega-6 fatty acids on the proliferation of mouse mammary tumor cells (MTC) was examined in a serum-free cell culture system. While the EGF-induced proliferation of normal mammary epithelial cells was shown to be enhanced by omega-3 and omega-6 fatty acids and prostaglandins (PGs), a majority (75-80%) of primary mammary tumors were not stimulated by these agents. Compared to normal cells, some MTC cultures showed a higher susceptibility to inhibition by omega-3 fatty acids. The general lack of response of MTC cultures to PGE2 and cyclic adenosine monophosphate (cAMP) suggests some alterations in the cAMP-mediated pathway. However, the PGE2-induced cAMP levels and cAMP-dependent protein kinase (PKA) activities in the tumor cells were comparable to normal cells. We conclude that the proliferation of mammary tumor cells either follow a cAMP-PKA-independent pathway or have some alterations in the serine/threonine kinase mediated signaling pathway.

Keratinocyte growth factor and acidic fibroblast growth factor are mitogens for primary cultures of mammary epithelium.

Mammary epithelial cells derived from the entire mammary parenchyma or only end buds were isolated by collagenase digestion of mammary glands from virgin mice. Cells were cultured within collagen gels in serum-free medium containing insulin. Keratinocyte growth factor (KGF or FGF-7) and acidic fibroblast growth factor (aFGF or FGF-1) stimulated multifold proliferation when added alone to this medium. Growth occurred as three-dimensional colonies within the collagen gel matrix. KGF stimulated growth was unaffected by adding heparin. Conversely, multifold growth stimulation by acidic FGF required heparin. Since end buds are the actively proliferating cell population of ductal glands, organ cultures of these structures were prepared. KGF stimulated 3H-thymidine incorporation in these end buds in the absence and presence of epidermal growth factor. These data suggest that acidic FGF and KGF may represent in vivo stromal factors capable of regulating mammary gland development.

Role of polyunsaturated fatty acids as signal transducers: amplification of signals from growth factor receptors by fatty acids in mammary epithelial cells.

The growth, morphogenesis and differentiation of milk producing epithelial tissues in the developing mammary glands require interaction with extracellular matrices and stimulation by hormones, growth factors and essential fatty acids. In primary culture, the proliferation of mammary epithelial cells (MEC), induced by epidermal growth factor (EGF), is enhanced and sustained by linoleate and its eicosanoid metabolites. Since a combination of linoleic acid (18:2 omega 6) and prostaglandin E2 or cAMP has synergistic effect on EGF-stimulated growth, it is suggested that additional cAMP-dependent protein kinase A (PK-A) independent pathways may also contribute to the linoleate effect on EGF action. Possible involvement of Ca2+/phospholipid-dependent protein kinase C (PK-C) is explored. Both linoleate and arachidonate can activate Type-II and Type-III protein kinase-C in MEC and a PK-C inhibitor can block growth stimulation by EGF and fatty acids. Like 12-O-Tetradecanoly phorbol-13-acetate (TPA), a PK-C activator which also enhances EGF-stimulated growth of MEC, linoleate can phosphorylate a 40-42 KD protein. EGF itself can stimulate transient phosphorylation of the same protein in MEC cultures but when supplemented with linoleate, which does not influence the ligand binding affinity of EGF-receptors, the transient phosphorylation signal in 40-42 KD protein is sustained.(ABSTRACT TRUNCATED AT 250 WORDS)

Pregnancy-dependent to ovarian-independent progression in mammary tumors delineated in primary culture: changes in signal transduction, growth factor regulation, and matrix interaction.

A model for the evolution of hormone-independent tumors from a pregnancy-dependent precursor is exemplified by the TPDMT-4[pregnancy-dependent (PD)] and T4OI96 [ovarian-independent (OI)] in vivo tumor lines developed in DDD mice. In vivo, the OI tumor grows rapidly in virgin mice and is more anaplastic than the PD tumor which, in virgin mice, grows as a hyperplastic alveolar gland from which tumors arise only during pregnancy. The regulation of the proliferation of these two tumors was compared in primary culture using a three-dimensional, serum-free, collagen gel cell culture system. In medium containing insulin, the growth of cell organoids from PD tumors was stimulated by the same factors that stimulate the growth of normal mammary epithelial cells from virgin or pregnant mice. These factors include progesterone and prolactin (but not estrogen), epidermal growth factor, basic fibroblast growth factor, linoleic acid, cyclic AMP, prostaglandin E2, phosphatidic acid, and lithium. Most of these tumors (about 80%) could not grow in medium with only insulin present; of those that did, growth was slow and was stimulated further by the above agents. PD tumor cells formed stellate colonies in the collagen matrix similar to those of normal cells. These findings show that the growth regulation of PD tumor cells is, in all aspects examined, similar to that of normal cells. In addition, the capacity for growth in the presence of only insulin (more autonomous growth) is not necessarily accompanied by deletions in responses to growth factors or hormones, or in lipid response pathways. In contrast, organoids from OI tumors needed only insulin for growth. The growth of some of these tumors was stimulated further by only basic fibroblast growth factor and phosphatidic acid. Cyclic AMP and agents that elevate intracellular cyclic AMP inhibited growth, the opposite of the response seen in normal or PD cells. OI organoids grew as cell masses rather than as stellate structures. These data show that while PD tumors are remarkably similar to normal cells in the regulation of their proliferation, OI tumors have incurred multiple defects in growth-regulatory pathways possibly including the production of autocrine growth factor(s). In addition, the ability of OI tumor cells to adhere to the collagen gel matrix and undergo normal morphogenesis is reduced. These results may highlight specific events required for the progression from ovarian dependency to ovarian independency related to the hormonal regulation of growth.

Casein accumulation in mouse mammary epithelial cells after growth stimulated by different hormonal and nonhormonal agents.

Mammary epithelial cells obtained from virgin mice were cultured in collagen gel with linoleic acid-containing serum-free growth medium supplemented with hormonal (PRL and progesterone, epidermal growth factor, somatomedin-C) or nonhormonal (lithium ion, phosphatidic acid containing phospholipid liposomes) growth stimulating agents. The phenotypes of the resulting progeny cells were compared by examining the ultrastructure, immunohistochemical staining for luminal epithelial and myoepithelial cells and casein, and assessing the quantity of biochemically detectable alpha- and beta-casein. Although there are some differences in ultrastructure and immunostaining in the progeny cell populations induced by different growth-promoting agents, all the cultures were able to accumulate alpha- and beta-casein on subsequent stimulation by PRL and linoleic acid in the second phase of culture. Since, in vivo, luminal epithelial cells of the mammary gland are the only cell type capable of synthesizing milk products, these results indicate that all the different growth stimulants, hormonal and nonhormonal, result in the predominant proliferation of luminal-type epithelial cells. These results have important implications for studies of the mechanism of growth control in and transformation of mammary epithelial cells.

Phospholipids containing polyunsaturated fatty acyl groups are mitogenic for normal mouse mammary epithelial cells in serum-free primary cell culture.

Epithelial cells obtained by collagenase digestion of mammary glands from virgin BALB/c mice were cultured in collagen gels in serum-free basal medium containing insulin (10 micrograms/ml), to which lipids or growth factors were added. Synthetic phospholipids were added as liposomes. Dilinoleoyl phosphatidic acid or phosphatidylserine or epidermal growth factor stimulated multifold growth. The optimum mitogenic effect of the phospholipids was dependent upon the presence of a polyunsaturated fatty acid esterified to the sn-2 position of the glycerol moiety. Dilinoleoyl phosphatidylcholine also stimulated growth but was generally less stimulatory than phosphatidylserine or phosphatidic acid, and phosphatidylethanolamine did not stimulate growth. Studies using phospholipids radiolabeled in either the sn-2 fatty acyl group or the glycerol backbone showed that the relative effect of phospholipids on growth did not correlate directly with the extent of their incorporation into cellular lipid, indicating that phospholipid turnover was the more important determinant for mitogenesis. Analysis of phosphatidic acid-stimulated growth suggested that both cAMP-dependent and cAMP-independent pathways were involved. Thus, mitogenic phospholipids stimulate proliferation by activating (directly or indirectly) multiple growth-regulatory pathways in mammary epithelial cells.

Basic fibroblast growth factor stimulates the growth and inhibits casein accumulation in mouse mammary epithelial cells in vitro.

The effects of basic fibroblast growth factor (bFGF) on the growth and differentiation of mouse mammary epithelial cells in serum-free collagen gel culture were examined. Epithelial cells obtained from virgin or midpregnant mice grew when bFGF was added to medium containing either insulin at a concentration greater than or equal to 1 microgram/ml or somatomedin-C (Sm-C) at 150 ng/ml. This growth-promoting effect is of the same magnitude as, and additive with, the growth-promoting effect of epidermal growth factor (EGF) or mammogenic hormones. The sensitivity of the cells to EGF or mammogenic hormones was not altered by exposure to bFGF. The progeny cells resulting from growth stimulation by bFGF are capable of accumulating casein upon subsequent stimulation by prolactin (PRL), but accumulate less casein than cells grown in response to EGF. bFGF also appears to reduce casein accumulation if it is added to the cultures at the same time as PRL.

Docosahexaenoic acid is neither synthesized nor retroconverted by the normal and tumor mouse mammary epithelial cells in primary culture.

Metabolism of 1-14C-[18:3(n-3)] and 1-14C-[22:6(n-3)] were investigated in the primary cultures of normal and tumor mouse mammary epithelial cells. Analysis of endogenous fatty acid composition indicated a decreased proportion of total (n-6) PUFA in the cultured tumor cells compared to normal cells. These cells can synthesize significant amount of 20:5 (n-3) and 22:5 (n-3) but not 22:6 (n-3), from 18:3 (n-3). There was very little or no retroconversion of 22:6 (n-3) by these cells. It has been concluded that mammary epithelial cells may be deficient in 4-desaturase activity and also that exogenous 22:6 (n-3), instead of serving as a source of 20:5 (n-3), may actually counter the effects of both 20:4 (n-6) and 20:5 (n-3) in the mammary tissue.